Ichthyophthirius multifiliis is a holotrichous ciliated protozoan which is an obligate parasite of freshwater fish. The life cycle of the parasite includes a free-living infectious stage (the theront or tomite) and an obligate fish-associated feeding stage (the trophont or trophozoite). The infective theront invades the skin and the gill epithelia, resulting in disturbances in respiratory and excretory functions. Once in epithelial tissue, the theront differentiates into the fish-associated feeding form known as a trophont. When the trophont is mature, it is released from the surface of the fish and secretes material forming a gelatinous cyst. Within the cyst, the mature trophont undergoes multiple cell divisions to produce hundreds of theronts, which are then released from the matrix to begin a new cycle of infection.
Protection from Disease Caused by I. multifiliis 
Ichthyophthiriasis, the disease caused by this parasite, is commonly referred to as “Ich” or “white spot disease.” Under conditions of intensive aquaculture, Ich frequently has a high morbidity and mortality, resulting in significant financial losses to fish producers.
Treatments are available for Ich-infected fish. However, the chemical treatments are effective only against the free-living theronts; there is no known agent for eliminating trophonts associated with the host. Furthermore, some of the chemotherapeutic agents used to treat Ich are suspected to leave residues in treated fish and to be carcinogenic. As a result, certain of the available treatments, e.g., malachite green, are not permitted for fish raised for human consumption. In addition, chemical treatments result in physiological stress to the infected fish beyond that resulting directly from the infection.
Those fish which survive infection by I. multifiliis are generally immune to further infection by the live parasite. Early reports suggested that fish (mirror carp) were successfully immunized by exposure to sublethal doses of the parasite (Hines et al., J. Fish. Biol. 6:373–378 (1974)) and by exposure to the live parasite in conjunction with chemical treatment. There have also been reports of at least partial protective immunity in fish vaccinated using the killed parasite. For example, a substantial decrease in the number of infective parasites on the body surface of goldfish which had been previously injected with killed theronts was observed when the fish were challenged with a measured dose of live theronts (Parker, Studies on the Natural History of Ichthyophthirius multifiliis Fouquet 1876, an Ectoparasitic Ciliate of Fish, Ph.D. Dissertation, The University of Maryland, College Park, Md. (1965)). Areerat (“The Immune Response of Channel Catfish, Ictalurus punctatus (Rafinesque), to Ichthyophthirius multifiliis”, unpublished Master's thesis, Auburn University) reported that channel catfish injected with formalin-fixed trophonts were protected when challenged with a lethal dose of infective theronts. Goven et al. (J. Fish Biol. 17:311–316 (1980)) reported initial protection against lethal infection when fish were injected intraperitoneally with theront cilia. The experiment was discontinued when all control fish had died and the fate of the vaccinated fish was not followed further.
More recently, however, Burkhardt et al. (J. Fish Dis. 13:401–410 (1990)) reported that neither immersion exposure nor intraperitoneal injection with killed I. multifiliis theronts conferred protective immunity to challenge doses of live theronts, although there was a delay in mortality of the vaccinated fish was observed. Likewise, intraperitoneal injection with theront cilia preparations did not prevent mortality, only delayed it. Only intraperitoneal injection with live theronts was effective in preventing mortality after challenge with infective parasites. Those fish which had been injected with live theronts remained protected against infection for an extended time, as evidenced by their resistance to challenge infections at 3 and 13 months after the original injection. Attempted immunization with formalin-fixed trophonts led to some delay in mortality but had an unclear effect on ultimate mortality.
The mucus coating of an immune fish participates in protection from Ich infection. Hines et al. (J. Fish. Biol. 6:373–378 (1974)) showed that both sera and mucus from immune fish was capable of immobilizing the infective form of I. multifiliis. These authors also noted that fish recovering from Ich had a different distribution of the parasite than did newly infected fish. Newly infected fish exhibited parasites all over the body while a recovering fish exhibits parasites primarily at edges of the fish. These are the parts of the fish which are least well supplied with blood, and therefore, would be less well supplied with antibodies.
Clark et al. (Devel. Comp. Immun. 1–2:581–594 (1988)) studied the sera of channel catfish that had been rendered immune to further Ich infection by exposure to sublethal surface infection and treated with chemotherapy. The sera of these immune catfish contain antibodies which specifically bind to I. multifiliis cilia; little cross-reactivity was observed for cilia prepared from the free-living ciliate Tetrahymena thermophila. Whole I. multifiliis cilia and a ciliary membrane fraction gave similar reactions with the immune sera, but axoneme fractions showed little differential reaction in comparisons between immune and preimmune sera. However, attempts to identify the ciliary proteins with which the antibodies reacted using blots from SDS gel electropherograms were not successful. Sera from immune fish also immobilize the parasite in vitro, with an apparent positive correlation between specific antibodies and immobilization of theronts in vitro.
There were early reports that fish vaccinated with Tetrahymena pyriformis and with T. thermophila or with cilia prepared from Tetrahymena were protected from Ich infection (e.g., U.S. Pat. No. 4,309,416, Gratzek et al). It had been proposed that the ciliary membrane antigens from Tetrahymena showed cross-reactivity with those of I. multifiliis. However, more recent reports showed that attempted vaccination of channel catfish with T. thermophila Lwoff cilia did not protect the fish from subsequent challenge with I. multifiliis (Burkhardt et al., J. Fish Dis. 13:401–410 (1990)). It has been postulated that the previous cross-reactivity was due to the conserved axoneme proteins, rather than due to serologically related ciliary membrane proteins.
I. multifiliis i-antigens
A novel mechanism of humoral immunity against I. multifiliis was recently described. Rather than being killed on the host, a majority of parasites are forced to exit fish prematurely in response to antibody binding (M. Cross, J. Fish Dis., 15:497–505 (1992))(T. Clark et al., Parasitol. Today, 13:477–480 (1997)). While the precise mechanism underlying this phenomenon is unknown, the target antigens responsible for premature exit have been identified as a class of abundant surface membrane proteins known as immobilization antigens, or i-antigens (T. Clark et al., Annu. Rev. Fish Dis., 5:113–131 (1995)). Antibodies against these proteins rapidly immobilize cells in vitro.
I-antigens are common to a variety of hymenostomatid ciliates and have been intensively studied in Paramecium and Tetrahymena where their expression undergoes marked variation in response to environmental stimuli (F. Caron et al., Annu. Rev. Microbiol., 43:23–42 (1989); Smith et al., J. Protozool., 39:420–428 (1992)). Antigenic switching in these cells results from the differential expression of multiple i-antigen genes under defined sets of conditions and represents one of the most striking examples of antigenic shift in nature. To date, there is little evidence that this type of variation occurs in Ichthyophthirius; however, steady-state levels of i-antigen transcripts vary as much as 50 fold during transition from the host-associated trophont to the infective theront stage, and it is clear that the genes for these proteins are developmentally regulated through the parasite life cycle (T. Clark et al., Proc. Nat. Acad. Sci. USA, 89:6363–6367 (1992)). Furthermore, serotypic variants of the i-antigens have been described among geographic isolates of the parasite (H. Dickerson et al., J. Euk. Microbiol., 40:816–820 (1993)).
Although i-antigens have gained considerable attention with regard to their mode of expression, their biological function remains obscure. In Paramecium and Tetrahymena, i-antigens are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor, and in some cases, form a thick layer that coats the plasma and ciliary membranes (F. Caron et al., Annu. Rev. Microbiol., 43:23–42 (1989)). This has led to speculation that their primary function is to shield the cell membrane from environmental insult; indeed, this fits a general model for the role of GPI-anchored proteins in lower eukaryotes. The fact that cross-linking of i-antigens at the surface of Ichthyophthirius elicits a physiological response in the parasite also suggests that these proteins may play a role in transmembrane signaling (T. Clark et al., Parasitol. Today, 13:477–480 (1997)). Consistent with this idea, i-antigen antibodies trigger mucocyst discharge in both I. multifiliis (T. Clark et al., J. Fish Biol., 31(A):203–208 (1987)) and Tetrahymena thermophila (J. Alexander, Trans. Amer. Microsc. Soc., 86:421–427 (1967)), as well as trichocyst discharge in Paramecium ssp.
The cDNA sequence associated with a 48 kD i-antigen from an isolate of I. multifiliis (G1 isolate, serotype A) was reported by Clark et al. (Proc. Nat. Acad. Sci. USA, 89:6363–6367 (1992)); serotyping was reported by Dickerson et al. (Annu. Rev. Fish Dis. 6:107–120 (1996)). A recombinant subunit vaccine derived from this cDNA sequence was reported by He et al. (Aquaculture 158: 1–10 (1997)). This subunit vaccine was engineered as a recombinant glutathione sulfotransferase (GST) fusion with a 105 amino acid fragment of the protein that the researchers identified as a potential antigenic epitope, corresponding to one of several tandemly repetitive amino acid sequence domains identified by Clark et al. (Proc. Nat. Acad. Sci. USA, 89:6363–6367 (1992)). The nucleotide sequence encoding the fusion construct was chemically synthesized and used for expression of the recombinant peptide in bacteria. Two amino acid substitutions relative to the native sequence were required in order to provide restriction sites in the corresponding DNA; moreover, protozoan glutamine codons TAA and TAG (which function as stop codons in E. coli and other conventional protein expression systems) were replaced by the universal glutamine codons CAG or CAA in order to allow expression of the fusion construct in E. coli. The fusion vaccine gave weak protection against an undefined isolate of I. multifiliis; 50% of the vaccinated fish were heavily infected with I. multifiliis upon challenge with the live parasite, compared to 75% of control fish.
The 48 kD i-antigen protein has been isolated from cultures of I. multifiliis (Clark et al., Annu. Rev. Fish Dis. 5:113–131 (1995); Lin et al., J. Protozoology 39:457–463 (1992)). In addition, a 55 kD i-antigen protein has been isolated from cultures of I. multifiliis and affinity purified and used in studies on passive immunity (T. L. Lin et al., Inf. Immun. 64:4085–4090 (1996)). Mouse monoclonal antibodies raised against this protein were effective to immobilize G5 isolates of I. multifiliis. However, a native i-antigen protein would be very difficult to obtain from I. multifiliis in large quantity because this obligate parasite cannot be easily cultured.
It is clear that an inexpensive and effective vaccine against Ich would be of great benefit to the aquaculture industry.